Recently, a method for the enzymatic amplification of specific segments of DNA has been described. This method, known as polymerase chain reaction (PCR), is based on repeated cycles of denaturation of double-stranded DNA, followed by oligonucleotide primer annealing to the DNA template, and primer extension by a DNA polymerase (Mullis et al. U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki et al. 1985). The oligonucleotide primers used in PCR are designed to anneal to opposite strands of the DNA, and are positioned so that the DNA polymerasecatalyzed extension product of one primer can serve as the template strand for the other primer. The PCR amplification process results in the exponential increase of discrete DNA fragments whose length is defined by the 5' ends of the oligonucleotide primers.
Application of PCR to isolate and analyze a particular DNA region requires knowledge of the DNA sequences flanking the region of interest. This generally limits amplification to regions of known DNA sequence. In the absence of the necessary sequence information, PCR amplification of a target DNA fraction in a complex DNA population is likely to result in the amplification of non-target DNA. Although methods have been developed to help overcome this problem, they have met with only limited success or application. These technologies have been used primarily for cloning populations of differentially expressed genes, for analyzing differences between two complex genomes (target DNA population) after subtractive hybridization of two complex DNA (or cDNA) mixtures, and for preparation of an "equalized cDNA library" (i.e., a cDNA library containing an equal abundance of all the cloned genes).
For selective amplification of target DNA, the target DNA is usually purified from non-target DNA using labor intensive and generally unreproducible physical separation techniques such as hydroxyapatite chromatography (Timblin et al., 1990; Ko, 1990), a streptavidin-biotin interaction (Wang et al., 1991) or oligo (dT)-latex beads (Hara et al., 1993). Another technique, known as "Coincidence Sequence Cloning," permits the selective amplification of common sequences shared between two complex and partially coincident DNA mixtures (Brookes et al., 1991). However, this procedure requires an M13 phage cloning procedure to produce the single-stranded DNA, as well as a preparative gel electrophoresis step to purify the "coincident sequences" before initiating the PCR amplification.
Techniques such as "chemical cross-linking subtraction" (Hampson et al., 1992), "vectorette" adapter technology (Riley et al., 1990) and "representational difference analysis" (Lisitsyn et al., 1993) have been developed which eliminate the physical separation step, thereby simplifying the technology for selective target amplification. However, "chemical cross-linking subtraction" can only be applied to mRNA-cDNA subtraction procedures and "vectorette" adapter technology requires knowledge of partial sequence information (the inner primer sequence) of the target DNA to be amplified. The highest efficiency of selective amplification of target DNA has been achieved using the simple "representational difference analysis" technique. This method is based on differences in the efficiency of exponential amplification of target DNA and the linear amplification of non-target DNA through the use of a special adapter design.
In view of the problems and limitations associated with each of the amplification methods discussed above, there remains a need for a method of enhancing the specificity and sensitivity of target DNA amplification. Such a method has been described, in part, by Lukianov et al., 1994. Application of the subject invention during PCR efficiently suppresses non-target DNA amplification while allowing for the exponential amplification of target DNA sequences.